Cloned gene encoding for bacteriocin from pediococcus acidilactici

ABSTRACT

Isolation and identification of a gene encoding for a bacteriocin precursor in Pediococcus acidilactici, cloning of the gene in a vector plasmid and transformation to bacteria is described. The bacteriocin is particularly useful for inhibiting Listeria in food products.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of copending application Ser. No. 07/635,965, filedon Dec. 31, 1990, which is a continuation-in-part of Ser. No. 375,344,filed Jul. 3, 1989, which is a continuation-in-part of Ser. No. 012,619,filed Feb. 9, 1987, now U.S. Pat. No. 4,883,673, and acontinuation-in-part of application Ser. No. 514,102, filed Apr. 25,1990.

BACKGROUND OF THE INVENTION Summary of the Invention

The present invention relates to a sequenced gene encoding for abacteriocin in Pediococcus acidilactici and in particular to a gene thatis essential for the production of the functional bacteriocin, calledhereafter helper protein, and to the cloned gene in a vector which istransformed into a bacterium. In particular, the present inventionrelates to a sequenced gene encoding for a bacteriocin derived from aplasmid in Pediococcus acidilactici.

PRIOR ART

The pediococci are a diverse group of Gram-positive homofermentativelactic acid bacteria often found as saphrophytes on vegetable material(Gonzalez, C. F., and B. S. Kunka, Appl. Environ. Microbiol.53:2534-2538 (1987); and Mundt, J. O., W. G. Beattie, and F. R. Wieland,J. Bacteriol. 98:938-942 (1969)). Commercially, pediococci are used inthe fermentation of vegetables (Pederson, Bacteriol Rev. 13:225-232(1949) and meats (Smith, J. L., and S. A. Palumbo, J. Food Prot.46:997-1006 (1983)).

Some strains of P. pentosaceus, P. cerevisiae and P. acidilactici havebeen found to contain resident plasmids although the roles of most ofthese remain unknown (Gonzalez, C. F., and B. S. Kunka, Appl. Environ.Microbiol. 46:81-89 (1983); Graham, D. C., and L. L. McKay, Appl.Environ. Microbiol. 50:532-534 (1985); and Raccach, M., CRC Crit. Rev.Microbiol. 14:291-309 (1987)). The association of raffinose fermentationand plasmid DNA has been reported (Gonzalez, C. F., and B. S. Kunka,Appl. Environ. Microbiol. 51:105-109 (1986)), as has been the ability ofP. acidilactici to ferment sucrose (Gonzalez, C. F. and B. S. Kunka,Appl. Environ. Microbiol 53:2534-2538 (1987)). Moreover, there have beenseveral reports which associate the production of bacteriocins with hostplasmid DNA (Daeschel, M. A., and T. R. Klaenhammer, Appl. Environ.Microbiol. 51:1538-1541 (1985); Gonzalez, C. F., and B. S. Kunka, Appl.Environ. Microbiol. 53:2534-2538 (1987); Graham, D. C., and L. . McKay,Appl. Environ. Microbiol. 50:532-534 (1985); and Bhunia et al, J.Applied Bact. 65:261-268 (1988)). It was shown by Gonzalez, C. F. and B.S. Kunka (Appl. Environ. Microbiol. 53:2534-2538 (1987)) thatbacteriocin production was encoded by a 9.0 kbp plasmid pSRQ11 in P.acidilactici PAC1.0. Further work (Pucci, M. P., E. R. Vedamuthu, B. S.Kunka and P. A. Vandenbergh, Appl. Environ. Microbiol. 54:2349-2353(1988)) demonstrated that the bacteriocin of P. acidilactici PAC1.0 wasactive against a wide spectrum of gram positive lactic acid bacteria,and also against Listeria monocytogenes. This anti-listerial activitywas observed in broth and on agar plates, as well as in some dairyproducts. Inhibition of L. monocytogenes by this bacteriocin, PA-1, hasalso been noted in fermented semi-dry sausage (Berry, E. D., M. B.Liewen, R. W. Mandigo and R. W. Huthine, J. Food Protection 53, 194-197(1990)) and fresh meat (Nielsen, J. W., J. S. Dickson and J. D. Crouse,Appl. Environ. Microbiol. 56, 2142-2145 (1990)). The cloning of genesfor the production of the bacteriocin has not been described and thiswould be useful for producing bacteriocin in significant quantities ingenera unrelated to Pediococcus, or enhancing production in thepediococci.

Cloned Gram-positive genes for different unrelated proteins have beenshown to express in Escherichia coli (Gilmore, M. S., Curr. Top.Microbiol. Immunol. 118:219-234 (1985); Rogeson, J. P., R. G. Barletta,and R. Curtiss III, J. Bacteriol. 153:211-221 (1983); and Smorawinska,M., J. C. Hsu, J. B. Hansen, E. K. Jagusztyn-Krynicka, Y. H. Abiko, andR. Curtiss III, J. Bacteriol. 153:1095-1097 (1983)).

OBJECTS

It is therefore an object of the present invention to provide thesequenced gene for the bacteriocin and its essential helper protein(s),which are used as vectors that can be transferred to othermicroorganisms that contain the genetic information of these genes insuch a way that the functional bacteriocin is produced by these newhosts. Such microorganisms are particularly in the genera Lactococcus,Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia,Bacillus and yeasts. These and other objects will become increasinglyapparent by reference to the following description and the drawings.

IN THE DRAWINGS

FIG. 1 shows a restriction endonuclease site map of pSRQ11. P.acidilactici PAC1.0 plasmid pSRQ11 is 9.0 kbp and contains the gene forPA-1 bacteriocin.

FIGS. 2A and 2B show restriction endonuclease site maps of pSRQ11.1 andpSRQ11.2, respectively. Both plasmids are 14.8 kbp and containerythromycin resistance (ery) genes at the locations indicated. The E.coli origin of replication (ori) and the remaining part of thechloramphenicol resistance (cm1) gene are shown. Numbered triangles (Δ)indicate areas of each plasmid which had been subsequently deleted.

FIG. 3A shows a restriction endonuclease site map of pSRQ220. PlasmidpSRQ220 is 9.3 kbp and is a chimera of Escherichia coli plasmid pBR322and PAC1.0 plasmid pSRQ11 digested with EcoRI and SalI and ligatedtogether. The Escherichia coli origin of replication (ori) and theampicillin resistance (amp) gene are indicated. The 5.6 kbp EcoRI-SalIfragment is indicated by the open box.

FIG. 3B shows a physical map of the 5.6 kbp EcoRI-SalI fragment frompSRQ220. The horizontal arrows denote open reading frames discussedhereinafter (ORF 1, ORF 2, and ORF 3). The horizontal lines, indicatedby numbered triangles (Δ1, Δ2, and Δ3), represent three deletionspresent in plasmids pUR5204 (Δ1), pSRQ220.2 (Δ2), and pSRQ11.13 (Δ3),respectively.

FIGS. 4A to Q show the nucleotide sequence of the 5.6 kbp EcoRI-SalIinsert from pSRQ220. The derived amino acid sequences of ORF1, ORF2, andORF3 are also shown. The arrow indicates the start of the mature PA-1bacteriocin. The TAG termination codons are denoted with an asterisk(*).

FIG. 5A shows a coomassie stained 5-22% acrylamide SDS-PAGE gel ofpurified PA-1. a=66,000, b=45,000, c=36,000, d=29,000, e=24,000,f=20,100, g=14,200, h=6500 Daltons Standards a through g are MW-SDS-70L(Sigma), standard h is aprotinin (Sigma)

FIG. 5B shows an unstained gel overlayed with a lawn of Pediococcuspentosaceus FBB63 indicator cells. Inhibition zone (large arrow) isapparent 1=110,000, 2=84,000, 3=47,000, 4=33,000, 5=24,000, 6=16,000Daltons. Prestained standards (Biorad) were used.

GENERAL DESCRIPTION

The present invention relates to a nucleotide sequence as given in FIGS.4A to Q and derivatives thereof which produces a bacteriocin precursor.

The present invention further relates to a vector containing anucleotide sequence containing ORF 1 as decribed in FIGS. 4A to Qmaintained in a bacterium in which the nucleotide sequence is precededby a promoter system and followed by a terminator sequence bothfunctional in the bacterium and express a bacteriocin encoded by thenucleotide sequence in the bacterium.

The nucleotide sequence of the present invention can be maintained in avector which operates in various bacteria or yeasts. All that isrequired is that the microorganisms express the bacteriocin.

The DNA encoding the bacteriocin can be replicated by means of apolymerase chain reaction as described in Chemical Engineering News,pages 36-46, Oct. 1, 1990 and in other references. The appropriate 3'and 5' terminal regions of the DNA encoding the bacteriocin can be usedas primers defining the region to be replicated.

The gene segment is preferably derived from Pediococcus acidilacticiNRRL-B-18050 also known herein as PAC1.0, which is deposited with theNorthern Regional Research Laboratory in Peoria, Ill. under the BudapestTreaty. The genes involved in bacteriocin activity are carried on a 9.0kbp plasmid designated herein as pSRQ11. A DNA segment (SalI to EcoRI;5.6 kbp) is ligated in purified form in a vector plasmid pBR322 andcalled pSRQ220. This plasmid is transformed to Escherichia coliNRRL-B-18429 and deposited at the same depository under the BudapestTreaty.

U.S. Pat. No. 4,883,673 which is assigned to a common assignee describesthe isolation of a bacteriocin from Pediococcus acidilacticiNRRL-B-18050 which inhibits various bacteria. A plasmid in this strainwas disclosed to encode for the bacteriocin. The bacteriocin wasdescribed to be useful in foods to inhibit bacterial spoilage. U.S. Pat.No. 4,929,445, assigned to a common assignee, describes a method ofusing the bacteriocin to inhibit Listeria monocytogenes which produces asevere illness in humans. The plasmid pSRQ11 was described as the sourceof the bacteriocin. The usefulness of the bacteriocin is wellestablished.

SPECIFIC DESCRIPTION

The following Examples show the steps in sequencing the gene encodingfor the bacteriocin.

Bacterial strains and media. The bacterial strains used are listed inTable 1.

                  TABLE 1                                                         ______________________________________                                        Bacterial Strains and Plasmids                                                Strain or plasmid                                                                        Remarks.sup.a       Reference                                      ______________________________________                                        P. acidilactici                                                               PAC1.0     Contains 9.0 kbp PA-1 pediocin                                                                    (4)                                                       plasmid, pSRQ11                                                    PAC1.14    PAC1.0 derivative cured of                                                                        (4)                                                       pSRQ11                                                             P. pentosaceus                                                                FBB63C     Sensitive indicator strain                                                                        (4)                                                       for PA-1 pediocin                                                  E. coli                                                                       V850       Hypersensitivity to macrolide                                                                     (5)                                                       antibiotics                                                        V871       Tetracycline sensitive                                                                            (7)                                            2g4        Tetracycline sensitive                                                                            (8)                                                       Ampicillin sensitive                                               Plasmids                                                                      pBR322     Ap.sup.r, Tc.sup.r  (1)                                            pACYC184   Cm.sup.r, Tc.sup.r  (2)                                            pVA891     Em.sup.r            (6)                                            pSA3       Em.sup.r, Cm.sup.r, Tc.sup.r                                                                      (3)                                            pSRQ11     9.0 kbp PA-1 pediocin                                                                             (4)                                                       plasmid                                                            ______________________________________                                         .sup.a Ap, ampicillin; Cm, chloramphenicol; Em, erythromycin; r,              resistance, and Tc tetracycline.                                              (1) Bolivar, F., et al., Gene 2:95-113 (1977).                                (2) Chang, A.C.Y., et al., J. Bacteriol. 134: 1141-1156 (1978).               (3) Dao, My Lein, et al., Applied and Environmental Microbiology,             49:115-119 (Jan. 1985).                                                       (4) Gonzalez, Carlos F. et al., Applied and Environmental Microbiology,       53:2534-2538 (Oct. 1987).                                                     (5) Macrina, Francis L., et al., Gene, 19:345-353 (1982).                     (6) Macrina, Francis L., et al., Gene, 25:145-150 (1983).                     (7) Tobian, Janet Ash, et al., Journal of Bacteriology, 160:556-563 (Nov.     1984).                                                                        (8) Bachman, K., Ptashne M., and Gilbert, W. Proc. Natl. Acad, Sci.,          4147-4178(1976).                                                         

Pediococcus spp. were routinely maintained on MRS agar (DifcoLaboratories, Detroit, Mich.). Escherichia coli strains were routinelycarried on Lennox L agar (Gibco/BRL, Gaithersburg, Md.). Escherichiacoli strains were also grown on modified MRS agar (no citrate oracetate) or in M9 medium (Maniatis, T., E. F. Fritsch, and J. Sambrook,Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982)) supplemented with 1% yeast extract(Oxoid, Ltd., Basingstoke, Hampshire, U.K.) and 1% Hy Case™ (SheffieldProducts, Norwich, N.Y.) for bacteriocin assays. Selective antibioticconcentrations were as follows: ampicillin, 25 ug/ml; tetracycline, 10ug/ml; erythromycin, 50 ug/ml; and chloramphenicol, 25 ug/ml. Allantibiotics were purchased from Sigma Chemical Co., St. Louis, Mo.

Bacteriocin assays. Production of bacteriocin was assayed as previouslydescribed (Gonzalez, C. F., and B. S. Kunka, Appl Environ. Microbiol.53:2534-2538 (1987)). Strains were patched on MRS agar or modified MRSagar for Escherichia coli and incubated at 35° C. for 18 hours. Theplates were then overlaid with soft agar (0.8%) seeded with indicatorcells. Isolates which produced a clear, defined zone of inhibition wereconsidered as bacteriocin producers.

One arbitrary unit (AU) of bacteriocin was defined as 5 microliters ofthe highest dilution of culture supernatant yielding a definite zone ofgrowth inhibition on the indicator lawn. The titer was expressed as thereciprocal of the highest dilution showing inhibition.

Isolation and analysis of plasmid DNA. Covalently closed circularplasmid DNA was isolated from Escherichia coli by the method of Clewelland Helinski (Clewell, D. B., and D. R. Helinski, Biochemistry9:4428-4440 (1970)). Escherichia coli strains were screened for plasmidcontent as previously described (Macrina, F. L., J. A. Tobian, K. R.Jones, R. P. Evans, and D. B. Clewell, Gene 19:345-353 (1982)).Pediococcus plasmid DNA was obtained by a scaled up modification of theLeBlanc and Lee procedure (LeBlanc, D. J., and L. N. Lee, J. Bacteriol.140:1112-1115 (1979)) as described by Gonzalez and Kunka (Gonzalez, C.F., and B. S. Kunka, Appl. Environ Microbiol. 46:81-89 (1983)). PlasmidDNA and restriction endonuclease digests were analyzed by agarose gelelectrophoresis on 0.8% agarose (Bethesda Research Laboratories, Inc.,Gaithersburg, Md.) slab gels. Size standards were Escherichia coli V517(Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S.McCowen, Plasmid 1:417-420 (1978)) for undigested plasmid DNA andHindIII -digested bacteriophage lambda DNA (Bethesda ResearchLaboratories) for restriction endonuclease - cleaved plasmid DNA.

DNA enzymology. Restriction endonuclease digestions were performed inlow-, medium-, or high-salt buffers, as recommended by Maniatis et al.(Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular cloning: alaboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982)) . Restriction enzymes were obtained from Bethesda ResearchLaboratories. DNA ligation reactions were carried out with T4 DNA ligase(Bethesda Research Laboratories) at 4° C. for 18 hours according toconditions recommended by the manufacturer.

Bacterial transformations. Escherichia coli was transformed by the CaCl₂heat shock method (Molecular cloning: a laboratory manual Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982)) with cells harvestedat an optical density at 660 nm of 0.2 to 0.3.

Purification of PA-1. Cultural supernatant was neutralized to pH 6.0with sodium hydroxide prior to gel filtration. A 450 ml aliquot ofneutralized supernatant was applied to a 5 cm×55 cm column (Pharmacia)containing one liter of Spectra/Gel AcA 202 (Spectrum) gel filtrationresin which had been equilibrated with 0.05M2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0. Activity was elutedusing the same buffer. Active fractions were pooled and applied to a 2.5cm×90 cm CM-Sepharose column equilibrated with 0.05M MES, pH 6.0.Activity was eluted with a linear gradient to 0.05M MES containing 1Msodium chloride, pH 6.0. Active fractions were pooled and dialyzedagainst a 10 fold excess of water using 1000 Da molecular weight cut-offdialysis tubing (Spectra-Por 6, Spectrum). Dialysate volume was reduced12 fold by applying the dialysis tubing directly to solid 20 KDapolyethylene glycol (Carbowax, Union Carbide) and was then furtherreduced 3.5 fold by vacuum centrifugation (Speed-Vac, Savant).Concentrated PA-1 was applied to a 1.0 cm×25 cm C18 reversed-phasecolumn (Vydac) equilibrated with 0.1% aqueous trifluoroacetic acid.Activity was eluted with a linear gradient to 45% acetonitrile over 30minutes at 1.5 ml/min. Active fractions were determined by directlyspotting aliquots of column effluent on MRS plates overlaid with softagar containing indicator cells. Active fractions were dried by vacuumcentrifugation and stored at -20° C. Specific activity is defined as AUper milligram protein. Protein analyses were performed using the BCAprotein assay kit (Pierce) using directions supplied with the kit.

EXAMPLE 1

Restriction endonuclease map of pSRQ11. The genes involved inbacteriocin PA-1 activity were previously shown to be associated withthe presence of a 9.0 kilobase plasmid, designated pSRQ11 (Gonzalez, C.F., and B. S. Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)).Plasmid pSRQ11 was digested with a number of restriction endonucleasesto generate the restriction site map shown in FIG. 1. The plasmidcontained several unique sites including EcoRI, NdeI, XbaI, SalI, andSstI. Other restriction enzymes which cleaved the plasmid were ClaI,HindIII, PvuII, and EcoRV. The following restriction sites were notfound on pSRQ11: AvaI, BamHI, SphI, NruI, PstI, and BglII.

EXAMPLE 2

Expression of PA-1 bacteriocin in E. coli. Plasmid pSRQ11 was digestedwith EcoRI and cloned into the EcoRI site on plasmid pVA891 (Macrina, F.L., et al., Gene 25:145-150 (1983)), which contains an erythromycinresistance marker expressed in both Escherichia coli and streptococci.Recombinant plasmids were obtained with pSRQ11 inserted in bothorientations and were designated pSRQ11.1 and pSRQ11.2 as shown in FIG.2. These Escherichia coli strains were assayed for expression of thePA-1 bacteriocin as previously described (Gonzalez, C. F., and B. S.Kunka, Appl. Environ. Microbiol. 53:2534-2538 (1987)). The strains weregrown on modified MRS medium and overlaid with Pediococcus pentosaceusFBB63 indicator strain Escherichia coli strains containing pSRQ11.1 andpSRQ11.2 both produced zones of inhibition in the indicator lawn whilethe control Escherichia coli V850 strains showed no zone of inhibition(Table 2).

                  TABLE 2                                                         ______________________________________                                        Plasmids derived from pSRQ 11                                                                                    Bacteriocin                                Name    Fragment         Vector    Activity                                   ______________________________________                                        pSRQ11.1                                                                              EcoRI nicked pSRQ11                                                                            pVA891    +                                          pSRQ11.2                                                                              EcoRI nicked pSRQ11                                                                            pVA891    +                                                  (opposite orientation                                                         from pSRQ11.1)                                                        pSRQ11.11                                                                             SalI deletion of pSRQ11.1                                                                      pVA891    +                                          pSRQ11.12                                                                             PvuII deletion of                                                                              pVA891    -                                                  pSRQ11.1                                                              pSRQ11.13                                                                             PvuII deletion of                                                                              pVA891    -                                                  pSRQ11.1                                                              pSRQ11.21                                                                             SalI deletion of pVA891    -                                                  pSRQ11.2                                                              pSRQ11.22                                                                             PvuII deletion of                                                                              pVA891    -                                                  pSRQ11.2                                                              pSRQ161 EcoRI nicked pSRQ11                                                                            pSA3      +                                          pSRQ210 3.7 kbp XbaI-SalI                                                                              pACYC184  -                                          pSRQ211 2.7 kbp HindIII  pACYC184  -                                                  fragment C                                                            pSRQ220 5.6 kbp EcoRI-SalI                                                                             pBR322    +                                          pSRQ220.1                                                                             ClaI deletion of pSRQ220                                                                       pBR322    -                                          pSRQ220.2                                                                             HindIII deletion of                                                                            PBR322    -                                                  pSRQ220                                                               pSRQ220.3                                                                             PvuII deletion of                                                                              pBR322    -                                                  pSRQ220                                                               pSRQ221 pACYC184 in XbaI site                                                                          pBR322    -                                                  of pSRQ220                                                            pSRQ221.1                                                                             XbaI deletion of pSRQ221                                                                       pBR322    +                                          pSRQ222 pACY184 XbaI-EcoRI                                                                             pBR322    -                                                  fragment in pSRQ220                                                   pUR5204 1.3 kbp HindIII-SalI                                                                           pBR322    -                                                  deletion derivative of                                                        pSRQ220                                                               pUR5205 pSRQ220 derivative with                                                                        pBR322    -                                                  disrupted HindIII site in                                                     ORF 3                                                                 pUR5206 pSRQ220 derivative with                                                                        pBR322    +                                                  disrupted HindIII site in                                                     ORF 2                                                                 pUR5217 pSRQ220 derivative with                                                                        pBR322    -                                                  BamHI linker insertion                                                        in BalI site of ORF 1                                                 ______________________________________                                    

The plasmid pSRQ11 was also cloned in the unique EcoRI site of the E.coli-Streptococcus shuttle plasmid pSA3. The resulting clone was calledpSRQ161. When the E. coli V850 strain carrying pSRQ161 (Table 2) wasgrown overnight in M9 medium supplemented with 1% yeast extract and 1%Hy Case, the filter sterilized culture supernatant yielded approximately400 AU/ml of the bacteriocin PA-1. This observation indicated that E.coli V850 (pSRQ161) was producing and excreting PA-1 into the media.Also, other E. coli strains were transformed with the plasmid pSRQ161and observed to produce PA-1. From this data, it was concluded that agene fragment encoding bacteriocin PA-1 from P. acidilactici PAC 1.0 canbe expressed and is functional in an E. coli host strain.

EXAMPLE 3

Deletion derivative analysis of pSRQ11 subclones

In order to localize the region encoding the PA-1 gene(s), SalI andPvuII deletion derivatives of pSRQ11.1 and pSRQ11.2 were obtained (FIG.2). The SalI deletion of pSRQ11.1 retained activity while the PvuIIdeletion derivatives displayed no zones of inhibition against theindicator strain (Table 2). Both the PvuII and SalI deletion derivativesof pSRQ11.2 expressed no PA-1 activity (Table 2). These data suggestedthat the bacteriocin gene was located on the approximately 5.6 kbpEcoRI-SalI fragment of pSRQ11.1 as shown in FIG. 2A. This 5.6 kbpEcoRI-SalI fragment then was subcloned into the EcoRI and SalIrestriction sites on the Escherichia coli plasmid pBR322 (Bolivar etal., Gene 2:95-113 (1977)), and the resulting chimeric plasmid wasdesignated pSRQ220 (FIG. 3A). The Escherichia coli strain containingpSRQ220 was assayed and found to express bacteriocin activity. Twoadditional deletion derivatives of pSRQ220, i.e., a plasmid derivativelacking a 2.7 kbp HindIII fragment and a plasmid derivative lacking a1.3 kbp HindIII-SalI fragment (FIG. 3B), were assayed and both found tobe negative for PA-1 activity. Also the following deletion derivativeswere obtained pSRQ210, which consisted of the pSRQ11, XbaI-SalI fragmentcloned into E. coli vector pACYC184 (Chang, A. C. Y., et al. J.Bacteriol. 134:1141-1156 (1978)), and pSRQ211, which consisted of pSRQ11HindIII fragment c (from map coordinates 1.5 to 4.2, FIG. 1) also clonedinto pACYC184. Neither of these two strains expressed PA-1 activity.Together with the bacteriocin PA-1 negative PvuII and ClaI deletionderivatives (FIGS. 2A, and 3B (Table 2)), these results show thatseveral genes, or one very long gene (or operon), present on the 5.6 kbpEcoRI-SalI fragment, are responsible for PA-1 activity.

EXAMPLE 4

Insertional inactivation of bacteriocin PA-1 production. Since the XbaIrestriction site is unique on both pSRQ11 and pSRQ220 and lies withinthe region involved in PA-1 production, it was chosen as a site toinsert a foreign DNA fragment and interrupt transcription of thebacteriocin gene. Plasmid pACYC184, approximately 4 kbp in size and alsocontaining a single XbaI site, was cloned into the XbaI site on pSRQ220.The strain containing the resulting recombinant plasmid, pSRQ221, wasassayed for PA-1 activity and proved negative (Table 2). When thepACYC184 insert was removed by XbaI digestion, followed by religation,resulting in pSRQ 221.1, activity was once again restored. Anotherconstruct where the XbaI-EcoRI fragment of pSRQ220 was replaced by theXbaI-EcoRI fragment of pACYC184 also was negative for bacteriocinactivity (Table 2).

EXAMPLE 5

Nucleotide sequence analysis of pSRQ220.

The DNA sequence of the 5.6 kbp SalI-EcoRI DNA fragment, as present onplasmid pSRQ220, was established by the Sanger dideoxy chain terminationprocedure (Sanger, F., Nicklen, S., and Coulson, A. R., Proc. Natl.Acad. Sci. USA, 74:5463-3967 (1977)) with the modifications as describedby Biggin et al (Biggin, M. D. et al., Proc. Natl. Acad. Sci USA,80:3963-3965 (1983)), using alpha-³⁵ S-dATP (2000 Ci/mmol) and Klenowenzyme (Amersham), ddNTP's (Pharmacia-PL Biochemicals) and dNTP's(Boehringer). The sequencing reaction products were separated on adenaturing polyacrylamide gel with a buffer gradient as described byBiggin et al. (Biggin, M. D. et al., Proc. Natl. Acad. Sci USA,80:3963-3965 (1983)). Purified, double-stranded plasmid DNA of pSRQ220served as template in the sequence reaction, following the proceduredescribed by Hattori and Sakaki (Hattori, M., and Sakaki, Y., Anal.Biochem. 152:232-238 (1986)). Deoxy-oligonucleotide primers weresynthesized on a DNA-synthesizer (Applied Biosystems 380A) using thePhosphoamidit technique (Barone, A. D. et al., Nucleic Acid Research,12:4051-4061 (1984)).

The DNA sequence when translated in all possible reading frames revealedat least three open reading frames (FIGS. 4A to Q). The first openreading frame (ORF 1) encodes a protein which consists of 62 amino acidresidues followed by a TAG stop codon (FIGS. 4A to Q). The second openreading frame (ORF 2), positioned just downstream of ORF 1, codes for aprotein which consists of 112 amino acid residues followed by a TAG stopcodon (FIGS. 4A to Q). Further downstream the third open reading frame(ORF 3) predicts a protein consisting of 724 amino acid residues with aTAG stop codon (FIGS. 4A to Q).

ORF 1 encodes a protein of 62 amino acids of which amino acid residues19 to 62 correspond entirely with the amino acid sequence of a protein,which was isolated from P. acidilactici NRRL-B-18050 called bacteriocinPA-1, and which, when separated on a polyacrylamide gel, inhibited P.pentosaceus FBB-63 effectively in an overlay experiment which is thesubject of U.S. application Ser. No. 514,102 (FIG. 4, and FIG. 5). Thisproves that ORF 1 encodes a precursor of bacteriocin PA-1, containing an18 amino acid N-terminal peptide which is cleaved off during the processof synthesis or excretion.

Both the PvuII deletion derivative pSRQ11.13 and the HindIII deletionderivative pSRQ220.2 (Table 2; FIG. 3B) result in a loss of PA-1bacteriocin activity. As these deletions disturb both ORF 2 and ORF 3,or ORF 3 only, but not the PA-1 bacteriocin encoding gene (ORF 1), itcan be concluded that also the presence of either ORF 2 or ORF 3, orboth is necessary for PA-1 bacteriocin activity.

EXAMPLE 6

Site-specific mutagenesis of genes involved in PA-1 bacteriocinproduction. The specific role in PA-1 bacteriocin production of each ofthe open reading frames was determined by introduction of frameshiftmutations in the various genes.

Plasmid pSRQ220 contains two sites for the restriction enzyme BalI. Oneis situated in the pBR332-part of the plasmid, whereas the other ispositioned within ORF 1 which encodes the PA-1 bacteriocin (FIG. 3A, and3B). A frameshift mutation in ORF 1 was introduced by insertion of adouble-stranded oligonucleotide linker fragment with the sequence5'-TGCATGGATCCTGATC-3' into this BalI-site. Plasmid pSRQ220 wastherefore partially digested with BalI, generating linear blunt-endedDNA molecules. This was achieved by incubation of the plasmid DNA in arestriction buffer for a short time period using only low amounts of therestriction enzyme. The linker fragment was added and allowed to ligatewith the BalI-treated vector DNA. Insertion of the linker fragmentdisrupts the BalI site, but introduces a new and unique BamHI site intothe plasmid, that was used for identification of the desired mutant.After transformation of the ligation mixture, plasmid DNA was isolatedfrom the transformants and screened for the presence of a BamHI site,concomitant with the loss of a BalI site. In this way plasmid pUR5217was identified which carried the desired linker insertion within ORF 1.Introduction of the mutation was confirmed by determination of thenucleotide sequence around the restriction site of the mutant. E. colicells containing pUR5217 were assayed for PA-1 bacteriocin activity andfound to have lost this property. This result is in good agreement withthe previous obtained deletion data and it again proves that thepresence of ORF 1 is essential for PA-1 activity. Restriction enzymeHindIII has only two restriction sites in pSRQ220, one of which ispositioned in ORF 2, while the other is positioned in ORF 3 (FIG. 3B).These sites were therefore well suited for introduction of mutations inthese genes. Plasmid pSRQ220 was partially digested with HindIII, asdescribed above. To fill in the 3'-restriction ends Klenow enzyme and amixture of the four dNTP's (A, T, G, C, 1 mM each) were added to theDNA-sample, followed by incubation at 37° C. for 30 minutes. Afterligation for 16 hours at 15° C. the DNA-mixture was transformed to E.coli 294. Plasmid DNA was isolated from the transformants and screenedfor the loss of the HindIII restriction sites by digesting with HindIII.Introduction of the mutations was confirmed by determination of thenucleotide sequence around the restriction site of each mutant. In thisway plasmid pUR5206 which carried a mutation at the HindIII site in ORF2, and plasmid pUR5205 which carried a mutation at the HindIII site inORF 3 were identified. E. coli cells containing pUR5206 were assayed andfound to express PA- 1 bacteriocin activity, whereas E. coli cellscontaining pUR5205 were negative for PA-1 bacteriocin activity. Fromthese data it can be concluded that, besides the presence of the PA-1bacteriocin gene (ORF 1), also the presence of an intact ORF 3 is neededfor PA-1 bacteriocin activity. The function of ORF 2 is not known.Although E. coli cells containing pUR5206 are able to producebacteriocin PA-1 activity, it cannot be ruled out that ORF 2 is involvedin the secretion or processing of bacteriocin PA-1. From the nucleotidesequence analysis some other tentative open reading frames can bededuced (data not shown). Therefore it is possible that otherinformation is present on the 5.6 kbp EcoRI-SalI fragment which is alsoneeded for PA-1 bacteriocin activity.

It is intended that the foregoing description be only illustrative ofthe present invention and the present invention is limited only by thehereinafter appended claims.

    __________________________________________________________________________    (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (vi) CURRENT APPLICATION DATA:                                                (A) APPLICATION NUMBER:                                                       (B) FILING DATE:                                                              (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5595                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Plasmid DNA                                               (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                          (v) FRAGMENT TYPE: N-terminal, internal and C-terminal                        fragments                                                                     (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pediococcus acidilactici                                        (B) STRAIN: NRRL-B-18050                                                      (C) INDIVIDUAL ISOLATE: PACl.0                                                (D) DEVELOPMENTAL STAGE: N/A                                                  (E) HAPLOTYPE: N/A                                                            (F) TISSUE TYPE: N/A                                                          (G) CELL TYPE: N/A                                                            (H) CELL LINE: N/A                                                             (I) ORGANISM: N/A                                                            (vii) IMMEDIATE SOURCE: N/A                                                   (viii) POSITION IN GENOME: N/A                                                (ix) FEATURE:                                                                 (A) NAME/KEY: bacteriocin encoding DNA                                        (B) LOCATION: ECORI to SalI                                                   DNA fragment 5.6 kbp.                                                         (C) IDENTIFICATION METHOD: sequencing                                         (D) OTHER INFORMATION: DNA needed for bacteriocin                             expression.                                                                   ( x) PUBLICATION INFORMATION: N/A                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCGACCGGAAATGATCTTTTTAACATCCAAGATAAAGAAAGCAAAATAGCTAAACAGAA60                GATTGTTAAATCTGGTAGTAATAAAGATGGCATACACACAAATAGAGCTATTAAACGCTG120               GTGGAAATTCTGGTAAAAGTTAATG TAAGCCTTAAGGTTTCAACTAAAGCAATTACAGTC180              AACCATAACCATAGTATTGGATTGTCATTTTATTGGCTATAAAATAGTAAATCAGTGAAT240               TTCATTACAAAAGGGCTCACAAAAAATTGTTTTCTTCCTCCAACAATAGCGAGACGCTTT300               TCTAATTG CTTGACCCAAAGAGCAATAGAATATTTTGAAGGTCCAAATTATTCTGTTAAT360              GATTTAAGTGAACGGCCTTCTTGGTGAAATTTAACCAATGAATCTTTGAAATCTTGTGAA420               TAACGAATTGACATAAAAATGCTCCTATATTTTCATTTTACGGACTGAATAAAAATA GTC480              CATTTTTTTAGTATAAGAGCAGTAAAACCAGACGTGGAAACCACGTGGTCTTTTAGTTGA540               TTCAGTAAAAGAAGCCGAAACCAACGTTTTCACGTTGGTTTCGGCTTCTTTGGCTTTTAA600               TTGCGGGAACGCACACAAAGAGCCAAAAAAGATTTGATAA AATCAAAGCTAGAAACTAGC660              TCCGGTCATGCTTGTTGCGATCATTATCGCGTAAGTCTTCTACGTGGGCATCACCACTCG720               TATCGATATCTAGTTCTTCGCGGCCGACGTTTTCACTTACTTGTTTCATATCTTCGTGTT780               CTTGTTTACGAATGTTAACTTC TTCTCGAACGACCGGGCGTTTGTTGACATCGGTAGTTG840              CAGCCGCACCATCTCCGGGCTTTCTTTCGATCACGATTTCTTCTCGTTTAAAATGAATAT900               ATAAACTGTGTCATAACTTAAAAGATACTGCGTTGATAGCCAGGTTTCAAAAATTGACCA960               AGATC GTTAACCAGTTTTGGTGCGAAAATATCTAACTAATACTTGACATTTAAATTGAGT1020             GGGAACTAGAATAAGCGCGTATTAAGGATAATTTAAGAAGAAGGAGATTTTTGTGATG1078                 Met                                                                          AAAAAAATTGAAAAATTAACTGAAAAAGAAATGGCCAATATCATTGGT1126                          LysLysIleGluLysLeuThrGluLysGluMetAlaAsnIleIleGly                              15-10-5                                                                       GGT AAATACTACGGTAATGGGGTTACTTGTGGCAAACATTCCTGCTCT1174                         GlyLysTyrTyrGlyAsnGlyValThrCysGlyLysHisSerCysSer                              151015                                                                        G TTGACTGGGGTAAGGCTACCACTTGCATAATCAATAATGGAGCTATG1222                         ValAspTrpGlyLysAlaThrThrCysIleIleAsnAsnGlyAlaMet                              202530                                                                        GCA TGGGCTACTGGTGGACATCAAGGTAATCATAAATGC1261                                  AlaTrpAlaThrGlyGlyHisGlnGlyAsnHisLysCys                                       3540                                                                          TAGCATTATGCTGAGCTGGCATCAATAAAGGGGTGATT TTATGAATAAGACTAAG1316                  MetAsnLysThrLys                                                               15                                                                            TCGGAACATATTAAACAACAAGCTTTGGAC TTATTTACTAGGCTACAG1364                         SerGluHisIleLysGlnGlnAlaLeuAspLeuPheThrArgLeuGln                              101520                                                                        TTTTTACTACAGAAGCACGATACTATCGAACC TTACCAGTACGTTTTA1412                         PheLeuLeuGlnLysHisAspThrIleGluProTyrGlnTyrValLeu                              253035                                                                        GATATTCTGGAGACTGGTATCAGTAAAACTAAACAT AACCAGCAAACG1460                         AspIleLeuGluThrGlyIleSerLysThrLysHisAsnGlnGlnThr                              404550                                                                        CCTGAACGACAAGCTCGTGTAGTCTACAACAAGATTGCCAGCCA AGCG1508                         ProGluArgGlnAlaArgValValTyrAsnLysIleAlaSerGlnAla                              556065                                                                        TTAGTAGATAAGTTACATTTTACTGCCGAAGAAAACAAAGTTCTAGCA1556                           LeuValAspLysLeuHisPheThrAlaGluGluAsnLysValLeuAla                             70758085                                                                      GCCATCAATGAATTGGCGCATTCTCAAAAAGGGTGGGGCGAGTTTAAC1604                          AlaIleAsnGluLeuAlaHisSerGlnLysGlyTrpGlyGluPheAsn                              9095100                                                                       ATGCTAGATACTACCAATACGTGGCCTAGCCAATAGTACTGATAAAGGGGATA1657                      MetLeuAspThrThrAsnThrTrpProSerGln                                            105110                                                                        TTGTAGTTGTCTAAGAAATTTTGGTCAAATATCTTTTTAGCATTAGGCGTCTTTCTTGCT1717              TTTGCAGGAGTTGCTACCATATCGGTGAGTGCTGACAGTT CCGCTACTATAGAATCAAAT1777             ACTAGCTCGAAAATCATCGATGGTGCAACTTATGAAGAAAACATCAGGGGCGTTATTCCT1837              ATTACGCTAACTCAATATTTGCATAAAGCTCAAACTGGAGAAAAATTTATTGTCTTTGTC1897              GGGTTCAAGGAGTGTGTGCATT GTCGTAAATTTTCTCCAGTCATGAAACAGTACTTACAA1957             CAAAGTCAGCATCCCATTTATTACTTAGACTATGGGAACAACGGGTCTTTCAGCATGGCT2017              TCTCAAAAACAAATAACTGATTTCTATTCAACTTTTGCAACCCCCATGAGTTTTATGGGA2077              ACGCC AACTGTTGCCTTGCTCGATAATGGTAAGGTGGTATCAATGACCGCTGGTGATGAT2137             ACCACTTTATCTGATTTACAACAGATTACTGCTGATTACAATAATCAGTAGTCACCTGGT2197              TAATATGGTTTTGTAACCAATGTAAAAGGCGATGGATCTTTGAAATCGTCTTTT TTTATG2257             CACAAATTTTAAAGATCGGTGGTTTGCTTATGTGGACTCAAAAATGGCACAAA2310                     MetTrpThrGlnLysTrpHisLys                                                      15                                                                            TATTATACAGCACAAGTTGATGAAAATGACTGTGGTTTAGCTGCACTA2358                          TyrTyrThrAlaGlnValAspGluAsnAspCysGlyLeuAlaAlaLeu                              101520                                                                        AATATGATC CTAAAATACTATGGCTCCGATTACATGTTGGCCCATCTT2406                         AsnMetIleLeuLysTyrTyrGlySerAspTyrMetLeuAlaHisLeu                              25303540                                                                      CGACAGC TTGCCAAAACAACTGCTGACGGTACAACTGTTTTGGGGCTT2454                         ArgGlnLeuAlaLysThrThrAlaAspGlyThrThrValLeuGlyLeu                              455055                                                                        GTTAAAGCA GCAAAACACTTAAATTTAAATGCCGAAGCTGTGCGTGCT2502                         ValLysAlaAlaLysHisLeuAsnLeuAsnAlaGluAlaValArgAla                              606570                                                                        GATATGGATGCTT TGACAGCCTCACAATTGCCATTACCAGTCATTGTT2550                         AspMetAspAlaLeuThrAlaSerGlnLeuProLeuProValIleVal                              758085                                                                        CATGTATTCAAGAAAAATAAG TTACCACACTACTATGTTGTCTATCAG2598                         HisValPheLysLysAsnLysLeuProHisTyrTyrValValTyrGln                              9095100                                                                       GTAACTGAAAACGATTTAATTATTGGTGAT CCTGATCCAACCGTTAAA2646                         ValThrGluAsnAspLeuIleIleGlyAspProAspProThrValLys                              105110115120                                                                  ACCACTAAAATATCGAAATCACAATTTG CTAAAGAATGGACCCAGATT2694                         ThrThrLysIleSerLysSerGlnPheAlaLysGluTrpThrGlnIle                              125130135                                                                     GCAATTATCATAGCCCCAACAGTTAAATA TAAACCCATAAAAGAATCA2742                         AlaIleIleIleAlaProThrValLysTyrLysProIleLysGluSer                              140145150                                                                     CGGCACACATTAATTGATCTAGTGCCTTTATTG ATTAAACAAAAAAGA2790                         ArgHisThrLeuIleAspLeuValProLeuLeuIleLysGlnLysArg                              155160165                                                                     TTAATTGGACTAATTATTACCGCAGCAGCTATAACAACA TTAATCAGT2838                         LeuIleGlyLeuIleIleThrAlaAlaAlaIleThrThrLeuIleSer                              170175180                                                                     ATTGCTGGTGCATATTTCTTTCAGTTAATTATCGATACTTATTTGCCG 2886                         IleAlaGlyAlaTyrPhePheGlnLeuIleIleAspThrTyrLeuPro                              185190195200                                                                  CACTTGATGACTAATAGGCTTTCACTAGTTGCCATTGGTCTGATTGT A2934                         HisLeuMetThrAsnArgLeuSerLeuValAlaIleGlyLeuIleVal                              205210215                                                                     GCTTATGCTTTCCAAGCAATTATCAACTATATACAAAGTTTTTTTACG 2982                         AlaTyrAlaPheGlnAlaIleIleAsnTyrIleGlnSerPhePheThr                              220225230                                                                     ATTGTATTAGGACAACGTCTCATGATCGACATCGTTTTAAAATACGTT30 30                         IleValLeuGlyGlnArgLeuMetIleAspIleValLeuLysTyrVal                              235240245                                                                     CACCATCTTTTTGATTTACCAATGAATTTTTTTACTACCCGTCATGTC3078                          HisH isLeuPheAspLeuProMetAsnPhePheThrThrArgHisVal                             250255260                                                                     GGTGAAATGACCTCACGCTTTTCTGATGCAAGCAAAATTATTGATGCA3126                          GlyGluMetThrSe rArgPheSerAspAlaSerLysIleIleAspAla                             265270275280                                                                  CTTGGAAGTACAACGCTCACCCTTTTTTTAGACATGTGGATTTTATTA3174                          LeuGlySerThr ThrLeuThrLeuPheLeuAspMetTrpIleLeuLeu                             285290295                                                                     GCAGTAGGGTTATTTTTGGCCTATCAAAACATCAATTTATTTTTATGC3222                          AlaValGlyLeu PheLeuAlaTyrGlnAsnIleAsnLeuPheLeuCys                             300305310                                                                     TCGTTAGTTGTGGTTCCAATTTACATCTCGATTGTTTGGCTATTTAAA3270                          SerLeuValValValP roIleTyrIleSerIleValTrpLeuPheLys                             315320325                                                                     AAAACTTTTAATCGTTTAAATCAAGATACAATGGAAAGCAATGCAGTT3318                          LysThrPheAsnArgLeuAsnGl nAspThrMetGluSerAsnAlaVal                             330335340                                                                     CTTAATTCTGCTATTATTGAAAGTCTCAGTGGCATAGAAACCATTAAA3366                          LeuAsnSerAlaIleIleGluSerLeuSerGly IleGluThrIleLys                             345350355360                                                                  TCACTAACTGGTGAAGCAACTACAAAAAAAAAGATTGACACACTATTT3414                          SerLeuThrGlyGluAlaThrThrLysLys LysIleAspThrLeuPhe                             365370375                                                                     TCTGACTTATTGCATAAAAACTTGGCTTATCAAAAAGCTGATCAAGGA3462                          SerAspLeuLeuHisLysAsnLeuAlaTyrG lnLysAlaAspGlnGly                             380385390                                                                     CAACAAGCTATCAAAGCAGCTACTAAATTAATCCTAACTATTGTTATC3510                          GlnGlnAlaIleLysAlaAlaThrLysLeuIleLe uThrIleValIle                             395400405                                                                     CTTTGGTGGGGTACTTTTTTTGTTATGCGACACCAACTGTCTTTAGGT3558                          LeuTrpTrpGlyThrPhePheValMetArgHisGlnLeuSer LeuGly                             410415420                                                                     CAGCTGTTAACTTATAATGCTTTGCTCGCTTACTTCTTGACCCCATTA3606                          GlnLeuLeuThrTyrAsnAlaLeuLeuAlaTyrPheLeuThrProLeu                              425 430435440                                                                 GAAAATATTATTAATTTACAGCCTAAACTACAAGCTGCCAGAGTGGCT3654                          GluAsnIleIleAsnLeuGlnProLysLeuGlnAlaAlaArgValAla                               445450455                                                                    AATAATCGATTAAATGAGGTTTATCTAGTAGAGTCTGAATTTTCTAAA3702                          AsnAsnArgLeuAsnGluValTyrLeuValGluSerGluPheSerLys                               460465470                                                                    TCTAGGGAAATAACTGCTCTAGAGCAACTAAATGGTGATATTGAGGTT3750                          SerArgGluIleThrAlaLeuGluGlnLeuAsnGlyAspIleGluVal                               475480485                                                                    AATCATGTTAGTTTTAACTATGGCTATTGTTCTAATATACTTGAGGAT3798                          AsnHisValSerPheAsnTyrGlyTyrCysSerAsnIleLeuGluAsp                              490 495500                                                                    GTTTCTCTAACAATTCCACATCATCAGAAGATTACTATTGTAGGCATG3846                          ValSerLeuThrIleProHisHisGlnLysIleThrIleValGlyMet                              505510 515520                                                                 AGTGGTTCGGGGAAAACGACCCTAGCCAAGTTGCTAGTTGGTTTTTTT3894                          SerGlySerGlyLysThrThrLeuAlaLysLeuLeuValGlyPhePhe                              525 530535                                                                    GAGCCTCAAGAACAGCACGGTGAAATTCAGATTAATCATCACAATATA3942                          GluProGlnGluGlnHisGlyGluIleGlnIleAsnHisHisAsnIle                              540 545550                                                                    TCTGATATTAGTCGCACAATTTTACGCCAATATATTAATTATGTTCCT3990                          SerAspIleSerArgThrIleLeuArgGlnTyrIleAsnTyrValPro                              555560 565                                                                    CAAGAACCTTTCATTTTTTCGGGCTCTGTATTAGAAAATTTATTGTTA4038                          GlnGluProPheIlePheSerGlySerValLeuGluAsnLeuLeuLeu                              570575 580                                                                    GGTAGCCGTCCTGGAGTAACTCAACAAATGATTGATCAAGCTTGTTCC4086                          GlySerArgProGlyValThrGlnGlnMetIleAspGlnAlaCysSer                              585590595 600                                                                 TTTGCTGAAATCAAAACTGATATAGAAAATTTGCCTCAAGGTTATCAT4134                          PheAlaGluIleLysThrAspIleGluAsnLeuProGlnGlyTyrHis                              605610 615                                                                    ACTAGATTAAGTGAAAGTGGATTCAACTTATCTGGTGGGCAAAAACAG4182                          ThrArgLeuSerGluSerGlyPheAsnLeuSerGlyGlyGlnLysGln                              620625 630                                                                    CGGTTATCAATAGCTAGAGCATTATTGTCTCCGGCACAATGTTTCATT4230                          ArgLeuSerIleAlaArgAlaLeuLeuSerProAlaGlnCysPheIle                              635640645                                                                     TT TGACGAATCAACCAGTAATTTAGACACCATTACTGAACATAAAATA4278                         PheAspGluSerThrSerAsnLeuAspThrIleThrGluHisLysIle                              650655660                                                                     GTCTCTAAGCTA TTATTCATGAAAGACAAAACGATAATTTTTGTAGCA4326                         ValSerLysLeuLeuPheMetLysAspLysThrIleIlePheValAla                              665670675680                                                                  CATCGTCTC AATATTGCGTCTCAAACCGATAAAGTTGTCGTTCTTGAT4374                         HisArgLeuAsnIleAlaSerGlnThrAspLysValValValLeuAsp                              685690695                                                                     CATGGAAAGA TTGTTGAACAGGGATCACATCGACAATTGTTAAATTAT4422                         HisGlyLysIleValGluGlnGlySerHisArgGlnLeuLeuAsnTyr                              700705710                                                                     AATGGGTATTATGC ACGGTTAATTCATAATCAAGAATAGCCTGACAAGAACC4474                     AsnGlyTyrTyrAlaArgLeuIleHisAsnGlnGlu                                          715720                                                                        AGTCTGCTATTGATAGACTATTCTTGTCCGTGAAATCCTCGCGTATTTCCGTGAGGA GCA4534             TAGTATATTTAGCGATCTTCAAATTTTAAGTATATTGATTCATATGTTTATCCTCCTAAG4594              TTTGAGGACAAACCGGTACATGTTATAATACTTCTACCGGCTTGTCCGGTGTCTGGAGCA4654              TTACCACATCCTTTCTGGGATAGAGGTAATGCTCTTCTAA AGTGCGCTTAAATAACCATT4714             GCCAGTGGTTAATCAGTGCTTTAACATGTTGCGTAAGTCATTGAGGGTGTCGGATTCCAC4774              GGCCTCAATGACTTTTTTTGTGCCTTATAATTAAAGGTGTTAAAATACGTCGTAACTTAC4834              CACCATAAAGCAGTCCAATTAA TTTATTGACTTCTAAGTAAAATACCAGGAGTTTTGCTA4894             TGAGTTAACTATGATCCTGGGTGGTCACTAAAACATTCCTTAATTCAGGGTCTATAACTA4954              TCAAATCGCCCCTCAAAATCATTGTTAAAATAACCCCCAATATCTATAATGTAGATGTTG5014              GGGGT TATTTATTTTAATATTAAATAAATAACTTCTTCTATTTGTCATCAATACTAAACA5074             ATAATTTGTACAAAGTGATTATTTCTTCTAGTTCTTCACGCGATACATGATCGACAATAG5134              TTTCATCAGTGACATGTCTTGCCCGTAAATCTAAGGCTATGGTTTGATCTAATA ATACTT5194             TTCCATATACTGTTTGACTACTAGTTAGTCGATGATACATTGGAAAATTACGCTTGGTAC5254              TGCTAATTGGAGCCGCAATCGTCATGTTACTTGTCTGACAGACTAGATCATTGCTTAGCG5314              CAATGGCTGGTCGCTTATTCATCTGTTCATGACCACG GCTTGGATTAAAGTTAACATAAA5374             ATATATCACCTTGGCTTACCATTGAAGTTCATTACCTTCTGACTTTCCCCAATCAAGCTC5434              GTGATCCCTTTTCCCGTCATCTTGCCAATCCTTAAATAGTTCGTGAATATTGGTTGGGTT5494              CTTTTTTATTGGTGTTAAAA CAATTGATCCATTTTCAATGGTTATTGTCATATCTTGGTT5554             ATCATCTAATTTCAGTTGTTTAATAATTTGGCTAGGAATTC5595                             

We claim:
 1. A bacteriocin precursor that is essentially free of otherproteins and having an amino acid sequence as given in FIG. 4 thatcorresponds to ORF1, and modifications thereof that still have thecapability of being converted into an active bacteriocin.